[Inhibitory effect of ortho-aminoazotoluene on diethylnirtosamine hepatocarcinogenesis in suckling mice. Phenomenon and possible mechanism].

The modifying effect of the one compound on carcinogenicity of the other in the combined application is attributed usually to some changes in the carcinogen metabolism, i.e. its activation or inactivation. In this paper, when used separately, diethylnitrosamine (DENA) induced 4-6 times more neoplastic lesions in the liver of suckling mice than ortho-aminoazotoluene (OAT) did. However, after combined treatment with both carcinogens the total number of hepatic lesions was significantly lower than that in mice treated with DENA only. Similar effect was observed when OAT was administered 3 days before or 3 days after DENA injection. The observed protective effect is not mediated at metabolic level, because OAT has no effect on metabolism of DENA in mouse liver. Our findings can be unequivocally explained by the competition of the carcinogens for target protein molecules, presumably transcription factors, participating in hepatocyte differentiation, which differently interact with and are diversely impaired by different compounds.

[Increase of general toxic effects and decrease of hepatocarcinogenic effects of diethylnitrosamine administered to mice in conjunction with isopropanol].

It is considered that diethylnitrosamine (DENA) is metabolized mostly in the liver with producing highly reactive compounds alkylating cellular macromolecules and causing toxic and carcinogenic effects. However, competitive inhibition of cytochrome P450 2E1, which metabolizes DENA, by its other substrate, isopropanol, does not weaken, but enhances the toxicity of DENA effect on mice and reduces the number of preneoplastic nodules and liver tumors induced by it. In short-term tests for mutagenicity (in the Ames test and the SOS-hromotest) DENA causes moderate damage of DNA that reduces liver microsomal enzymes, that metabolize nitrosamines. The obtained data indicate the predominant role of the initial compound as compared to its metabolites in toxic, and probably hepatocarcinogenic, effects on DNA in mice.

[Expression of nuclear hormone receptors PPAR, LXR and RXR in the liver and lipid and glucose levels in blood in susceptible and resistant to hepatocarcinogenesis mice strains].

Earlier it was shown that male mice of the DD/He strain were highly susceptible to ortho-aminoasotoluene (OAT) induced hepatocarcinogenesis, and resistant to spontaneous liver tumor development as compared to the CC57BR/Mv strain. In the present work we have made a comparative investigation of peroxisome proliferator-activated receptor (PPAR), liver X-receptor (LXR) and retinoic X-receptor (RXR) mRNA levels in liver as well as concentrations of corticosterone, glucose, lipids and insulin in blood of male DD/He and CC57BR/Mv mice. Using the multiplex RT-PCR method it was found that PPAR-alpha, PPAR-gamma, RXR-alpha and RXR-beta mRNA content was essentially decreased in the liver of DD mice as compared to mice of the CC57BR strain. No significant interstrain differences of LXR-alpha and LXR-beta mRNA content were found. In DD micetere was more then the 3-fold decrease of blood content of corticosterone, which is involved in PPAR and RXR regulation. DD mice demonstrated a significant decrease in blood serum glucose and insulin concentrations as well as higher reactivity to insulin as compared with CC57BR mice. Elevated blood total cholesterol and cholesterol HDL level were found in DD mice whereas triglyceride content was basically the same in both mouse strains. It is known that glucocorticoids, PPAR and RXR play crucial role in transcription regulation of inflammation response. Therefore our data allow to suggest that decreased corticosterone level in blood, PPAR and RXR mRNA content in liver of the DD strain may lead to induction of inflammation by OAT exposure, resulting in a high incidence of tumorigenesis in this strain.

[O-aminoazotoluene inhibits DENA-induced hepatocarcinogenesis when used together in mice].

When used separately, diethylnitrosamine (DENA) initiates 4-6 times more neoplastic lesions in the liver of suckling mice than ortho-aminoazotoluene (OAT) lower. However, after combined treatment with either carcinogen the total number of hepatic lesions was significantly than that in mice injected with DENA only. Similar inhibition of DENA-induced hepatocarcinogenesis was observed when OAT was administered 8-12 hrs after DENA. Our findings cannot be adequately explained except by competition of the carcinogens for supposedly target molecules of protein origin, presumably, transcription factors, which contribute to generation of genetic information. They have different affinities for different compounds and, therefore, suffer different damage from the latter functioning.

[Effect of hepatocarcinogenicity of estragole on the glucocorticoid-mediated induction of liver-specific enzymes and the activity of the transcription factors FOXA and HNF4 in the liver of mouse and rat].

The carcinogenic effects of estragole in mice of the earlier unexplored strain ICR has been studied. It has been shown that there is a distinct correlation between the extent of inhibition of glucocorticoid-mediated induction of tyrosine aminotransferase and trypthophan oxygenase after acute administration of estragole and the frequency of liver tumors after estragole exposure. Estragole inhibits the induction of these enzymes only in female mice, but not in male mice and rats. DNA-binding activities of liver-enriched transcription factors were investigated on carcinogen-susceptible and -resistant animals. Estragole decreases the HNF4 (hepatic nuclear factor 4) and FOXA DNA-binding activities only in susceptible female mice, but not in nonsusceptible male mice and rats and does not influence the C/EBP and HNF1 activities. Pentachlorophenol, which prevents the hepatocarcinogenic effect of estragole, abolishes its inhibitory effect on tyrosine aminotransferase and trypthophan oxygenase glucocorticoid induction and restores the FOXA and HNF4 DNA-binding activities. The parallelism between the hepatocarcinogenic effects of estragole and the inhibition of FOXA and HNF4 DNA-binding activities serves as an additional argument for the involvement of these factors in the mechanisms of tumor suppression in the liver.

[Inhibitory effect of thyroxine on late stages of hepatocarcinogenesis induced in suckling mice by diethylnitrosamine].

5 groups of 14-day ICR male mice received intraperitoneally diethylnitrosamine in the dose 50 mcg/g body weight. After weaning, they were kept in standard laboratory conditions. When aged 6 months, the mice of the 1st group were sacrificed and the number of iron-negative premalignant nodules in their livers as well as adenomas in the lungs were recordered. Since then, mice of the 3rd and 5th groups received 0.0002% L-thyroxine as drinking water throughout the experiment. Control mice of the 2nd and 4th groups were given tap water. At 9 months of age, total number of hepatic nodules in mice of group 2 was slightly (40%) higher whereas the number of gross nodules (2-5 mm in diameter) and extremely gross ones (> 5 mm) were 3- and 6-fold higher than in mice of group 1. After additional 2 months, the number of tumor nodules in the liver of control mice of group 5 could not be counted because of their mixed growth. By contrast, in thyroxine-treated mice, the number of gross nodules, as well as total number of tumor nodules in the livers were not higher than those in control mice of group 1. The data obtained indicate that exogeneous thyroxine inhibits growth of liver tumors even at the late stages of carcinogenic process. In addition, it increases the food intake and relative weight of kidneys and decreases relative weight of testes but does affect growth of pulmonary adenomas in male mice under study.

[Mosaic expression of the lacZ reporter-gene under control of 5'-regulatory sequences of the alpha-S1-casein gene in transgenic mice].

Phenomenon of mosaic expression at cellular level is widely presented in tissues and organs of transgenic animals. The communication is concerned a study of the mosaics in transgenic mice carrying the lacZ reporter-gene under control of the bovine and goat alpha-S1-casein genes with 5'-flanked sequences of various ex-tent: pCLZ1--721bp, pCLZ2-- 2001 bp and pCLZ3 3409 bp constructs. Five transgenic founders were generated by injection of the recombinant DNA into zygotes: pCLZ 1 - N 16, pCLZ2 - N 37 and pCLZ3 N 7, N 36, and N 48. Positive for J3-galactosidase activity cells were detected in lactating mammary glands of all transgenic females, however, distribution of the positive cells was variable. We observed two types of mosaics: clonal or "lobule" type with positive cells filling the whole of the globule or stochastic type with single positive cells scattered over one or different lobules. Two types of mosaics were characteristic of all the transgenic animals, although, females carrying the pCLZ2 transgene showed "lobule" type more often than transgenic animals with the transgenes pCLZ and pCLZ3. It is suggested that the stochastic type of mosaics occurs in the cells at terminal stage of differentiation, whereas the <> type arises from positive for P-galactosidase proliferating precursors. Analysis of the inheritance of the transgenes in different lines demonstrated that the pCLZl transgene was inserted in the X-chromosome of the founder whereas the other two localized in autosomes. Localization of the pCLZl transgene in the X-chromosome did not influence the mosaicism; it was similar to that of transgenic animals carrying the transgenes in autosomes. Ectopic expression of the reporter-gene was detected in mandibular glands from the offsprings of the founders N 16 and N 37 only, as well as in atrezed follicles in N 37. The weak ectopic expression saggests that the 5 S-flanked regulatory sequences used in the constructs are able to provide perfect tissue-specific expression of the reporter-gene.

[Morphological characteristics of liver and white adipose tissue during development of melanocortin obesity in C57Bl/6J Agouti yellow mutant mice].

The changes of liver and white adipose tissue (WAT) morphology during development of melanocortin obesity in female Agouti yellow (genotype A Y/a) C57Bl/6J mice have been investigated. Mouse strain of a/a genotype was used as a control. Results have been compared with the hormonal and metabolic changes during development of obesity and type 2 diabete in Agouti yellow mice. The tissues obtained from mice of 8-, 11-, 15- and 22-week old have been analyzed. The morphology of liver and WAT of A(Y) and control animals did not differ during 15 weeks of age. 40% of A(Y) mice revealed liver steatosis (fatty liver) at the age of 22 weeks. In addition, elevation in the inflammatory and proliferating processes in the liver and severe inflammation in WAT has been observed in these animals. Since as early as 15 weeks old A(Y) mice demonstrated the appearance of insulin resistance characteristics we conclude that hormonal and metabolic abnormalities could play a role of the primary factor of pathological reorganization of liver and WAT morphology.

[Changes of lipid-carbohydrate metabolism during development of melanocortin obesity in the mice with the Agouti yellow mutation].

Melanocortin obesity develops after puberty in mice with Agouti yellow mutation. The aim of the work was to study dynamics of the main characteristics oflipidcarbohydrate metabolism during the obesity development. We used female mice of C57BI/6J strain homozygous for recessive mutation nonagouti (a/a-mice) who were predisposed to the obesity, and heterozygous for dominant mutation Agouti yellow (Ay-mice) who were not predisposed to the obesity. Food consumption and body weights were measured from week 4 to week 22. Some animals were decapitated on 8, 11, 13, 15, 22 weeks. Hyperphagia (6 week) proceeds to body weight increasing (7 week) in the Ay-mice. In the Ay-mice compared to a/a-mice, adiposyte size and blood level of leptin were increased on the 11th week, blood levels of fatty acids and glucose--on the 13th week, blood level of insulin--on the 15th week. The hyperphagia seems to promote development ofmelanocortin obesity, stable disturbances of lipid metabolism appearing before those in glucose metabolism. Complete metabolic obesity syndrome was formed after the 15th week of life.

[Effect of phenobarbital and thyroxine on hepatocarcinogenesis initiated in mice by nitrosoethylurea and diethylnitrosamine].

13-14-day old mice of ICR and CBA strains were given a single intraperitoneal injection of nitrosoethylurea (80 mg/kg) or diethylnitrosamine (50 mg/kg). 2 weeks later, they were given drinking water containing phenobarbital (1 g/L) or thyroxine (2 mg/L). The control mice were given only tap water. 29.4% of male and 42.1% of female ICR mice who had received nitrosoethylurea died of leukemia within 3-6 months after the carcinogen treatment. There was no case of leukemia in mice treated with diethylnitrosamine. Nitrosoethylurea induced 3-more often lung adenomas than diethylnitrosamine. Phenobarbital and thyroxine did not affect development of either leukemias or lung adenomas. By contrast, phenobarbital significantly elevated the number and size of hepatic lesions, whereas thyroxine markedly decreased them in all the experiments. The total and free thyroxine levels were significantly decreased in the blood of mice given phenobarbital and increased in mice given thyroxine. The data obtained indicate that thyroid hormones suppress tumor development in the mouse liver and that the promotion of hepatic tumoro-genesis by phenobarbital is presumably caused by the elimination of this suppressing effect of the thyroid hormones.

[Different responses of DD/He and CC57BR/Mv mice to fasting]. / Neodinakovaia reaktsiia na golodanie myshei linii DD/He i CC57/BR/Mv.

Reaction to fasting of 2 mice strains differing in their sensitivity to spontaneous and induced hepatocarcinogenesis, has been investigated. It was shown that mice of both strains displayed similar stress reaction after 3-day fasting manifested in increase in blood corticosterone level; and decrease in testosterone level. At the same time, both strains demonstrated opposite changes at tissue- and enzyme levels in the liver. It was shown that DD/He mice, highly sensitive to induction of liver tumors, were characterized by significant increase in tyrosine aminotransferase (TAT) activity and reduction of lipid droplets in hepatocytes. CC57BR/Mv mice, demonstrating high frequency of spontaneous hepatomas and insensitive to induction of such tumors, were characterized by a decrease in the TAT activity and fatty infiltration of the liver.

[Effect of cortisol administration and stress actions in early ontogeny on hormonal induction in adult rats]. / Vliianie vvedeniia kortizola i stressornykh vozdeistvii v rannem ontogeneze na gormonal'nuiu induktsiiu u vzroslykh krys.

The influence of cortisole injections and stress ("handling") in the early ontogenesis (during the first 9 or 16 days of life) on the process of thyrosine aminotransferase induction by cortisol in the adult rats has been studied. It was shown that both the injection of the hormone in the young rats and "handling" led to the manifested trace effects: (1) stable increase of thyrosine aminotransferase activity in the liver of adult (5-6 months) rats and (2) appearance of peculiar tolerance to cortisol in the form of decrease in the ability of cells to induce thyrosine aminotransferase in response to the hormone injection. The data obtained suggest that the sensitive period of postnatal ontogenesis when cortisol or "handling" exert such a stable effect is limited by the 3rd and 9th days after birth. The causes of such "biochemical imprinting" are considered with respect to the increased sensitivity of the genetical system of cells-targets to the transcription inducers during the early postnatal development.

[Mechanisms of seasonal changes in the circadian corticosteroid function of rats]. / Mekhanizmy sezonnykh izmenenii tsirkadnoi kortikosteroidnoi funktsii u krys

Data are presented concerning seasonal changes in the circadian 11-OCS rhythm in the adrenal glands in the plasma of rats. For the purpose of studying the mechanisms of control of circadian 11-OCS rhythms during the spring and autumn seasons there were studied circadian corticosteroid reactions to ACTH and stress. The "Cosinor" method of study was used for analysis of the real circadian rhythm. The data obtained permit to suppose that in autumn the rhythm of the adrenal glands was associated with the circadian rhythm of the central links of the hypothalamo-hypophyseo-adrenal system, and during the spring season an important contribution in the formation of the circadian rhythm of the adrenocortical activity was brought by the adrenal mechanisms.